Proteolysis is one of the most important and frequent enzymatic reactions that occur both within and outside of cells. Proteolysis results in the activation, degradation, and maturation of nascent polypeptides. These modifications ultimately influence cell cycle progression, cell-cell signaling, metabolism, and many other processes required for development and survival of multicellular organisms. Proteolytic enzymes, or proteases, catalyze the hydrolytic cleavage of peptide bonds. Proteases can be classified based on their enzymatic mechanism, substrate specificity, active site configuration, and overall three-dimensional structure. The mammalian serine proteases comprise one of the most well-characterized and versatile protease families. This family is exemplified by the pancreatic and plasma serine proteases.
Pancreatic serine proteases are secreted from the pancreas into the duodenum where they degrade proteins ingested in food. Examples of these proteases include chymotrypsin, trypsin, elastase, and pancreatic kallikrein. Plasma serine proteases, which include thrombin and Clr, are involved in blood coagulation and immune response. Thrombin converts fibrinogen, a large soluble plasma protein, into fibrin, a smaller insoluble protein that aggregates to form blood clots. Clr is a component of the complement system, a complex of proteins that perforates the cell membranes of invading microorganisms.
Most mammalian serine proteases are synthesized as zymogens, inactive precursors that are activated by protease cascades. For example, trypsinogen is converted to its active form, trypsin, by enteropeptidase. Enteropeptidase is an intestinal protease that removes an N-terminal fragment from trypsinogen. The remaining active fragment is trypsin, which in turn activates the precursors of the other pancreatic enzymes. Likewise, proteolysis of prothrombin, the precursor of thrombin, generates three separate polypeptide fragments. The N-terminal fragment is released while the other two fragments, which comprise active thrombin, remain associated through disulfide bonds.
The catalytic active site is conserved among mammalian serine proteases. A defining characteristic of the active site is the catalytic triad, the conserved asparagine, histidine, and serine residues critical for catalysis. These residues form a charge relay network that facilitates the binding of substrate to protease. In addition to active site residues, other residues of the protease form an oxyanion hole. These residues stabilize the substrate during catalysis and vary among different subclasses of serine proteases.
Other tissue-specific serine proteases have been discovered. In mouse, for example, the serine protease neuropsin is expressed primarily in the hippocampus, a region of the brain involved in long-term memory. Direct electrical stimulation of the hippocampus modulates neuropsin messenger RNA levels. Neuropsin cDNA predicts a 260-amino acid protein that shares 38% amino acid identity with pancreatic trypsin. Residues that form the catalytic triad and the oxyanion hole are conserved between the two proteins. A putative glycosylation site at asparagine 110 of neuropsin is contained within a unique consensus sequence, YNNSN, the same as that found in kallikrein-like serine proteases. Neuropsin may influence synapse formation and neuronal connectivity in the hippocampus in response to neural signaling. (Chen, Z.-L. et al. (1995) J. Neurosci. 15:5088-5097.)
Prostate-specific antigen (PSA) is a serine protease synthesized and secreted exclusively by epithelial cells in the prostate gland. PSA is most related to kallikrein-like serine proteases. Inactive PSA is a 262-amino acid, 35-kilodalton glycoprotein that contains a signal peptide sequence from amino acid residues 1-17 and an activation peptide from residues 18-24. Proteolytic removal of both these N-terminal peptides is required for activation of PSA. Ten cysteines in PSA form intramolecular disulfide bonds, and the catalytic triad is conserved. PSA is secreted into the seminal fluid in response to the male sex hormone, androgen. The substrate for PSA is seminogelin, a large protein that promotes the coagulation of seminal fluid. Degradation of seminogelin by PSA results in the liquefaction of seminal fluid which allows increased sperm motility. (Henttu, P. and Vihko, P. (1994) Ann. Med. 26:157-164; and Riegman, P. H. J. et al. (1989) Bioch. Biophys. Res. Commun. 159:95-102.)
Prostate cancer accounts for more than 13% of all cancer deaths and more than 28% of all cancers in men in the United States. These statistics are surpassed only by those reported for lung cancer. (McCance, K. L. and Huether, S. E. (1994) Pathophysiology: The Biological Basis for Disease in Children and Adults, Mosby-Year Book, Inc., St. Louis, Mo.) Normally, PSA is confined to the prostate, and only very low levels of PSA are detectable in serum. However, in the sera of patients with malignant prostate tumors, PSA levels are significantly elevated. The serum concentration of PSA generally increases with the severity of the clinical or pathological manifestations of the disease. Currently, serum PSA is the most sensitive physiological marker for monitoring the progression of prostate cancer and its response to therapy. Serum PSA measurements in conjunction with other diagnostic tools may have application in early detection of the disease. PSA can also identify the prostate as the origin of a metastatic tumor. (Brawer, M. K. and Lange, P. H. (1989) Urology 33:11-16; and Henttu and Vihko, supra.)
Interestingly, the increase in serum PSA in prostate cancer patients is not due to increased expression of the gene encoding PSA. In fact, PSA gene expression is decreased as a result of disease. The accumulation of PSA in the serum may be attributed either to the disruption of the integrity of the prostate epithelium, which would result in the leakage of prostate contents, or to the depolarized secretion of PSA from the prostate epithelium into areas accessible to the circulation. (Henttu and Vihko, supra.)
The discovery of a new prostate-associated serine protease and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of reproductive disorders and cancer.